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- Supporting Information
GPCR mRNA splicing with its resultant changes to receptor sequence adds considerable potential diversity to cellular responses by altering receptor pharmacology and signalling. The secretin family of GPCRs is only a small family of 15 members, yet their capacity to contribute to a diverse array of functions is expanded through distinct receptor splice variants and interactions with receptor activity-modifying proteins (RAMPs) (Hay et al., 2006; Furness et al., 2012). Different combinations of RAMPs and receptor splice variants could generate many unique phenotypes that fine-tune physiological responses. One receptor that exemplifies this is the calcitonin (CT) receptor (CTR).
CT is a 32-amino acid hormone that modulates calcium homeostasis through inhibiting osteoclast-mediated bone resorption (Sexton et al., 1999; Pham et al., 2005). CT is well-known to enhance bone stability and has been widely used for the treatment of bone disorders. For instance, salmon (s) CT (e.g. Miacalcin, Novartis, Basel, Switzerland) is an approved drug for treating osteoporosis. Its receptor, CTR, accommodates a seven-transmembrane helical structure with a long extracellular N terminus (∼150 amino acids), a prominent characteristic of the secretin family GPCRs. Several CTR splice variants have been described in humans, with others existing in different species (Furness et al., 2012). These include a truncated CTR variant isolated from human breast carcinoma MCF-7 cells (Albrandt et al., 1995). The receptor is the leucine 447 polymorphic insert negative variant human (h) CT(a), lacking the 16-amino acid insert in the first intracellular loop with an additional truncation of the first 47 amino acids in its N terminus [Δ(1–47)hCT(a)]. This truncation contains the predicted signal sequence of 22 amino acids and one of the four potential N-linked glycosylation sites (Ho et al., 1999). The N terminus of the secretin family GPCRs is the initial docking site for their peptide ligands and the extreme N terminus of CTR is thought to contain contact points for CT (Dong et al., 2004a,b; Pham et al., 2004; 2005). Tissue distribution analysis identified the transcript of this truncated hCT(a) variant in various human tissues, including kidney, skeletal muscle, lung, both the caudate nucleus and the hypothalamus regions of the brain, whole brain and fetal brain (Albrandt et al., 1995). It was also faintly visible in the pancreas but not in SK-N-MC neuroblastoma or U-2 OS osteogenic sarcoma cell lines.
CTR can also associate with RAMPs (RAMP1, RAMP2 or RAMP3) and act as pharmacologically distinct receptors for amylin (Amy): the AMY1, AMY2 and AMY3 receptors (Poyner et al., 2002). The abbreviation Amy denotes the peptide, but AMY denotes the receptor complex for this peptide. Amy and CT are related peptides, both belonging to the CT family of peptides, which also includes the calcitonin gene-related peptides (CGRPs), adrenomedullin (AM) and AM2. Interestingly, CGRP and Amy are equipotent at the AMY1 receptor; the physiological relevance of this receptor to the actions of either peptide is still unknown (Hay et al., 2005).
Amy was initially discovered in amyloid deposits of human insulinoma and the pancreas of type 2 diabetic patients (Cooper et al., 1987). This peptide inhibits gastric emptying, gastric acid secretion, postprandial glucagon secretion and food intake (Guidobono et al., 1994; Young et al., 1995; Fineman et al., 2002; Mack et al., 2007). SymlinTM (Amylin Pharmaceuticals, San Diego, CA, USA), a modified form of human Amy, is commercially available for the treatment of type 1 and type 2 diabetes.
Despite the physiological and therapeutic relevance of CT and Amy, how they interact with their receptors is still poorly understood and there is still much to be learnt about the potential contribution of CTR splice variants to their biology. In particular, very limited work has been carried out with the truncated hCT(a) variant and it is not known whether this receptor retains its ability to interact with RAMPs. Given that the extreme N terminus of the closely related receptor, calcitonin receptor-like receptor (CLR), is involved in RAMP1 interactions, CTR association with RAMPs could be reduced in the truncated CTR variant (Ittner et al., 2005; Haar et al., 2010). As RAMP association is needed for high affinity Amy binding, this CTR splice variant could act as a natural regulator of AMY receptor function in some cells. Therefore, we hypothesized that Δ(1–47)hCT(a) has different pharmacology from CTR due to changes in RAMP association and/or loss of peptide contact points. Distinct receptor phenotypes were observed depending on which RAMP Δ(1–47)hCT(a) was co-expressed with. These effects appeared to be independent of changes in RAMP expression.
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- Supporting Information
Signal peptides have been suggested to play a crucial role in the expression of family B GPCRs (Couvineau et al., 2004). However, removal of the first 47 residues in the N terminus, including the predicted signal peptide, did not substantially impact upon the cell-surface expression of Δ(1–47)hCT(a). In Cos7 cells, expression of Δ(1–47)hCT(a) was slightly higher than hCT(a), while in HEK293S cells, a modest reduction of ∼30% in expression was observed. It has previously been reported that the same CTR/RAMP complexes exhibit different signalling profiles in different cellular backgrounds, likely due to the expressed complement of proteins that is unique to each cell type (Morfis et al., 2008). Therefore, the difference in expression may be related to the differential cellular backgrounds between Cos7 and HEK293 cells. On this basis, the predicted signal peptide does not appear to play a major role in CTR.
Δ(1–47)hCT(a) gives the first insight into the mode of RAMP interaction with CTR. In Cos7 cells, there was a reduction in both mychRAMP1 and FLAGhRAMP1 translocation to the cell surface with Δ(1–47)hCT(a), compared with the full-length receptor, but this effect was much smaller (∼10% reduction) and was not statistically significant in HEK293S cells. Again, this difference observed in Cos7 versus HEK293 cells may reflect the individual properties of the two cell types. Therefore, it seems unlikely that the first 47 amino acids of CTR play a major role in associating with RAMP1. A previous study using mouse CLR and human PTH (parathyroid hormone) receptor chimeras showed that N terminal residues 23–60 of the closely related CLR were important for mouse CLR and mouse RAMP1 association (Ittner et al., 2005). The crystal structure of the extracellular domains of the human CLR/RAMP1 complex shows that the major interactions involve residues on the αC1 helix of hCLR spanning methionine 42 to glutamine 54 (Haar et al., 2010). Amino acid sequence alignment shows that methionine 42 of CLR corresponds to methionine 49 of hCTR, which is beyond the truncated region in Δ(1–47)hCT(a). Although there is no structure available for the extracellular domain of CTR, it is likely that CTR and CLR adopt a similar structure and that methionine 49 and beyond in CTR may be principally responsible for RAMP1 interactions in the AMY1(a) receptor.
Our data suggest that CTR may interact differently with RAMP2. While RAMP1 expression was unaffected with Δ(1–47)hCT(a) in HEK293S cells, RAMP2 translocation to the cell surface was reduced by approximately 50%. Residues 1–47 of CTR may be more important for RAMP2 than RAMP1 association. The recent publication of the crystal structure of the extracellular domains of the CLR/RAMP2 complex showed several differences between CLR/RAMP1 and CLR/RAMP2 complexes (Haar et al., 2010; Kusano et al., 2012). In particular, CLR/RAMP interfaces were suggested to be different between the two complexes, where RAMP2 is distinctly orientated from CLR compared with RAMP1. It is thus likely that differences in the protein to protein interface may also be expected in the CTR/RAMP complexes; some residues in regions 1–47 may be more critical to CTR/RAMP2 association compared with those in CTR/RAMP1 complexes. We were unable to compare effects with RAMP3 due to tag artefacts.
hCT potency was significantly reduced at Δ(1–47)hCT(a) compared with hCT(a) in both cell types, suggesting that the first 47 amino acids of the receptor contain a region or residue important for hCT interactions. This is consistent with a previous investigation where a photoaffinity hCT probe labelled residue T30 in the N terminus of hCT(a) (Dong et al., 2004a). The exact contribution of T30 of hCT(a) to its interaction with hCT is not clear; alanine substitution at this position showed normal binding and cAMP response to hCT, but a nearby residue may be important (Dong et al., 2004b). The potency of all other peptides, apart from sCT, was also reduced at Δ(1–47)hCT(a) compared with hCT(a) in both cell types. It is possible that this was due to a reduction in cell-surface expression in HEK293S cells. However, cell-surface expression was not reduced in Cos7 cells, suggesting that it is more likely that there may be one or more interaction sites within the first 47 amino acids of hCT(a) that are important for interactions with these peptides, or the truncation altered the receptor conformation, which, in turn, affected the peptide interactions. Both hCT(a) and Δ(1–47)hCT(a) displayed a potency order of sCT > hCT > rAmy > CGRP (Supporting Information Table S3). This is consistent with the initial characterization performed where both the full-length and the truncated hCT(a) showed a potency order of sCT > hCT > hAmy (Albrandt et al., 1995). In the previous study, Amy potency seemed to be reduced at Δ(1–47)hCT(a), which was consistent with our work. Unchanged sCT potency is also consistent between studies. On the other hand, we see a clear reduction in hCT potency in two cell backgrounds. Formerly, there was no reduction in hCT potency. Given that both studies have used Cos7 cells, the reason for this discrepancy is unclear.
sCT displayed high and equivalent potency at the truncated and full-length hCT(a). To further investigate this, we used two other sCT derivatives, sCT8–32, a peptide fragment of sCT, and AC187, a peptide made by replacing the last three amino acids of sCT8–32 with those from rAmy. Both peptides serve as antagonists at CT(a) and AMY1(a) receptors (Hay et al., 2005). Both peptides had lower affinity at Δ(1–47)hCT(a) compared with the full-length hCT(a) receptor. This apparently contradicts the full-length sCT data but sCT is a very potent/high affinity agonist that elicits poor reversibility of binding to human CTR; on the other hand, antagonists including CT8–32 bind reversibly to the receptor (Hilton et al., 2000; Poyner et al., 2002). Therefore, an effect of the CTR truncation may be difficult to detect for full-length sCT.
The differential peptide responses seen at the different AMY receptors versus hCT(a) were consistent with the previously published data (Hay et al., 2005). For example, rAmy, CGRP and hCT were approximately equipotent at hAMY1(a), while hCT was more potent than rAmy or CGRP at hCT(a). The formation of AMY receptor phenotype by Δ(1–47)hCT(a) was evident for all three RAMPs, despite variable reductions in RAMP1 or RAMP2 translocation to the cell surface. Peptide potencies for rAmy, hαCGRP, hβCGRP and Tyr°hαCGRP were all enhanced at Δ(1–47)hAMY1(a) compared with Δ(1–47)hCT(a), indicating that Δ(1–47)hCT(a) still associates with RAMP1 and forms a functional hAMY1(a) receptor. In addition, Δ(1–47)hAMY1(a) appeared to be fully functional at the cell surface, displaying equivalent peptide responses to the full-length receptor for all agonists tested except for hCT. In particular, Δ(1–47)hAMY1(a) exhibited a potent CGRP receptor phenotype, displaying a higher potency towards hαCGRP than rAmy. This potency was even greater than that elicited from full-length hAMY1(a) receptor and demonstrated altered peptide potency order (Supporting Information Table S3). Like RAMP1, the AMY receptor phenotype was observed for Δ(1–47)hAMY2(a) as peptide potencies of rAmy and hαCGRP were significantly enhanced in the presence of RAMP2. Δ(1–47)hAMY3(a) also displayed an AMY receptor pharmacology where both rAmy and hαCGRP potencies were significantly enhanced in the presence of RAMP3.
The phenotype of Δ(1–47)hCT(a) in the presence of RAMPs was particularly interesting. hαCGRP, hβCGRP and Tyr°hαCGRP potencies, which were reduced at Δ(1–47)hCT(a), were restored at Δ(1–47)hAMY1(a); all these agonists showed equivalent or even higher peptide potencies at Δ(1–47)hAMY1(a) compared with hAMY1(a). This effect was particularly clear for hαCGRP, which showed no loss of activity at Δ(1–47)hCT(a) in the presence of RAMP1 in Cos7 or HEK293S cells. It seems that the presence of RAMP1 essentially rescued the function of this receptor, with respect to CGRP and generated a receptor with a unique pharmacological profile. RAMP3 had a similar effect to RAMP1, where hαCGRP potency was also maintained at Δ(1–47)hAMY3(a) compared with hAMY3(a). Therefore, RAMP1 and RAMP3 may have a role in stabilizing some peptide interactions sites in the Δ(1–47)hCT(a) receptor, either by directly contributing peptide contacts or altering the receptor conformation. On the other hand, RAMP2 did not appear to play such a role in rescuing CGRP potency at Δ(1–47)hAMY2(a); hαCGRP potency was also reduced at Δ(1–47)hAMY2(a) compared with hAMY2(a). This is consistent with the pharmacology of receptor complexes, where RAMPs 1 and 3 tend to support higher affinity CGRP interactions than RAMP2 (Poyner et al., 2002; Gingell et al., 2010; Moore et al., 2010). rAmy potency was affected most in RAMP2 complexes. The overall magnitude of reduction in potency observed at the truncated receptors was greater for hAMY2(a) receptors compared with hAMY1(a) receptors. It is possible that these effects were related to reduction in expression of with Δ(1–47)hAMY2(a) at the cell surface.
Despite the changes seen in cAMP accumulation between Δ(1–47)hCT(a)/Δ(1–47)hAMY1(a) and full-length receptors, no differences in ERK1/2 phosphorylation were observed. The reason for this is not yet defined because the mechanisms for ERK regulation at these receptors are not well defined. This could reflect distinct receptor conformations with the removal of residues 1–47 disturbing the conformation, leading to cAMP accumulation but not that needed for ERK1/2 phosphorylation. It could also reflect non-stoichiometric regulation of ERK, such that a submaximal cAMP response is still compatible with maximal ERK1/2 phosphorylation. Each receptor splice variant, coupled with individual RAMPs, creates essentially new receptor units, each with potentially unique properties in terms of ligand interaction and signalling characteristics. More work is needed to determine the relevance of ERK1/2 phosphorylation versus cAMP to CT and Amy biology and in particular what the predominant signalling mechanism might be for Δ(1–47)hCT(a) expressed in native tissues. A cell-type heavily reliant on cAMP for CT function might be more affected by this receptor variant than one in which ERK1/2 is more significant.
The effect of the I347T mutation found in Δ(1–47)hCT(a) was also determined in this study. There was little effect in receptor expression with this mutation apart from an enhanced Δ(1–47)hCT(a) expression when isoleucine was present at position 347. Furthermore, Δ(1–47)hCT(a) receptor function was not affected both in the presence and in the absence of RAMP1. Therefore, the I347T mutation reported in the previous study (Albrandt et al., 1995) does not seem to have any major impact to (Δ(1–47)hCT(a) receptor expression or function. I347T could be a sequencing artefact.
In this study, a naturally occurring variant of hCT(a) containing a 1–47 truncation at the N terminus has been characterized. Despite lacking the first 47 amino acids in the N terminus, Δ(1–47)hCT(a) retains its ability to reach the cell surface. RAMP association is mostly retained and the Δ(1–47)AMY1(a) receptor is a super-potent CGRP receptor. On the other hand, Δ(1–47)hCT(a) loses functionality as a CT receptor, suggesting that residues 1–47 may either have a role in maintaining overall receptor structure or possibly contain one or more interaction sites for peptides. Thus, the functionality of the Δ(1–47)hCT(a) variant depends on whether it is co-expressed with RAMP and with which RAMP it is expressed. Δ(1–47)hCT(a) expression has been detected by Southern blot in a range of human tissues, including kidney, skeletal muscle, lung and hypothalamus (Albrandt et al., 1995). In kidney, this could potentially result in reduced CT responsiveness (Findlay and Sexton, 2004). On the other hand, CGRP responsiveness could be enhanced if RAMP1 were co-expressed with this variant. Unfortunately, there is no corresponding information on RAMP expression or Amy/CGRP function in these human tissues and so it is very difficult to speculate as to the physiological function of this variant. Nevertheless, the data presented here certainly indicate that where RAMPs are co-expressed with this CTR splice variant, the resulting receptors could contribute to the actions of Amy and CGRP. The potential contributions of such receptors to the physiology of these peptides should not be overlooked, although teasing out their individual roles will be a challenging task.